Method to isolate DNA

Abstract

An improved method for isolating DNA from biological samples is provided.

1. A method for isolating DNA from a biological sample comprising animal cells comprising the following sequential steps:
 (a) separating the cells comprising DNA from the remainder of the biological sample; 
 (b) contacting the separated cells comprising DNA of step (a) with a hypertonic, high salt reagent having a concentration of salt therein so as to form a suspension of said biological cells; 
 (c) contacting the suspension of step (b) with a lysis reagent so as to lyse the biological material containing DNA to form a lysate comprising DNA and non-DNA biological components of the biological material, wherein the hypertonic, high salt reagent in step (b) comprises salt in an amount effective to precipitate proteins out of the lysate; and 
 (d) separating the DNA from the non-DNA biological components of the lysate of step (c) to yield isolated DNA. 
 
     
     
       2. The method of  claim 1 , wherein the biological sample is selected from the group consisting of animal tissue, cultured animal cells, blood cells, and body fluids. 
     
     
       3. The method of  claim 1 , wherein the biological sample is a bone marrow sample. 
     
     
       4. The method of  claim 1 , wherein the biological sample is whole blood. 
     
     
       5. The method of  claim 1 , where the non-DNA biological component is selected from the group consisting of proteins, lipids, RNA, and carbohydrates. 
     
     
       6. The method of  claim 1 , wherein the salt is selected from the group consisting of soluble sodium, ammonium, or potassium salts. 
     
     
       7. The method of  claim 1 , wherein the concentration of the salt is greater than 1 M. 
     
     
       8. The method of  claim 1 , wherein the concentration of the salt is greater than 2 M. 
     
     
       9. The method of  claim 1 , wherein the lysis reagent comprises a detergent. 
     
     
       10. The method of  claim 1 , wherein the lysis reagent comprises an anionic detergent. 
     
     
       11. The method of  claim 10 , wherein the anionic detergent is chosen from the group consisting of sodium, potassium, and lithium salts of dodecyl sulfate. 
     
     
       12. The method of  claim 10 , wherein the concentration of the anionic detergent is greater than 0.1% w/v based on the volume of the lysis reagent. 
     
     
       13. The method of  claim 1 , wherein the lysis reagent further contains an RNase solution. 
     
     
       14. The method of  claim 1 , wherein the step of separating the DNA from the lysate further comprises physically precipitating non-DNA biological components from the lysate without the use of any additional reagents, to yield a non-DNA precipitate, and a solution containing DNA. 
     
     
       15. The method of  claim 14 , wherein the step of separating the DNA from the lysate further comprises centrifuging the lysate. 
     
     
       16. The method of  claim 15 , further comprising contacting said solution containing DNA with an alcohol to yield a precipitate comprising isolated DNA. 
     
     
       17. The method of  claim 16  further comprising contacting the isolated DNA with a wash solution. 
     
     
       18. The method of  claim 16 , wherein the isolated DNA is treated with a hydration reagent. 
     
     
       19. A method for isolating DNA from a biological sample comprising red blood cells and white blood cells comprising the following sequential steps:
 (a) contacting the biological sample with a red blood lysis reagent to lyse the red blood cells; 
 (b) separating the white blood cells from the lysed red blood cells; 
 (c) contacting the white blood cells with a hypertonic, high-salt reagent having a concentration of salt therein to suspend the white blood cells in a solution of said hypertonic, high-salt reagent; 
 (d) subsequently contacting the white blood cells of step (c) with a lysis reagent so as to lyse the biological material containing DNA to form a lysate containing DNA and non-DNA cellular material, wherein the hypertonic, high salt reagent in step (c) comprises salt in an amount effective to precipitate proteins out of the lysate; and 
 (e) separating the DNA from non-DNA cellular material of the lysate to yield isolated DNA. 
 
     
     
       20. The method of  claim 19 , wherein the biological sample is selected from the group consisting of blood cells and body fluids. 
     
     
       21. The method of  claim 19 , wherein the biological sample is a bone marrow sample. 
     
     
       22. The method of  claim 19 , wherein the biological sample is whole blood. 
     
     
       23. The method of  claim 19 , where the non-DNA biological component is selected from the group consisting of proteins, lipids, RNA, and carbohydrates. 
     
     
       24. The method of  claim 19 , wherein the salt is selected from the group consisting of soluble sodium, ammonium, or potassium salts. 
     
     
       25. The method of  claim 19 , wherein the concentration of the salt is greater than 1 M. 
     
     
       26. The method of  claim 19 , wherein the concentration of the salt is greater than 2 M. 
     
     
       27. The method of  claim 19 , wherein the lysis reagent comprises a detergent. 
     
     
       28. The method of  claim 19 , wherein the lysis reagent comprises an anionic detergent. 
     
     
       29. The method of  claim 28 , wherein the anionic detergent is chosen from the group consisting of sodium, potassium, and lithium salts of dodecyl sulfate. 
     
     
       30. The method of  claim 28 , wherein the concentration of the anionic detergent is greater than 0.1% w/v based on the volume of the lysis reagent. 
     
     
       31. The method of  claim 19 , wherein the lysis reagent further contains an RNase solution. 
     
     
       32. The method of  claim 19 , wherein the step of separating the DNA from the lysate further comprises physically precipitating non-DNA biological components from the lysate without the use of any additional reagents, to yield a non-DNA precipitate, and a solution containing DNA. 
     
     
       33. The method of  claim 32 , wherein the step of separating the DNA from the lysate further comprises centrifuging the lysate. 
     
     
       34. The method of  claim 32 , further comprising contacting said solution containing DNA with an alcohol to yield a precipitate comprising isolated DNA. 
     
     
       35. The method of  claim 34  further comprising contacting the isolated DNA with a wash solution. 
     
     
       36. The method of  claim 34 , wherein the isolated DNA is treated with a hydration reagent. 
     
     
       37. A method for isolating DNA from a biological sample comprising microbial cells comprising the following sequential steps:
 (a) separating the cells comprising DNA from the remainder of the biological sample; 
 (b) contacting the separated cells comprising DNA of step (a) with a hypertonic, high salt reagent having a concentration of salt therein so as to form a suspension of said biological cells; 
 (c) contacting the suspension of step (b) with a lysis reagent so as to lyse the biological material containing DNA to form a lysate comprising DNA and non-DNA biological components of the biological material, wherein the hypertonic, high salt reagent in step (b) comprises salt in an amount effective to precipitate proteins out of the lysate; and 
 (d) separating the DNA from the non-DNA biological components of the lysate of step (c) to yield isolated DNA. 
 
     
     
       38. The method of  claim 37 , wherein said microbial cells are bacterial cells.